Enhanced probe binding

ABSTRACT

Methods for enhancing the binding of oligonucleotide probes to DNA and RNA are disclosed. The methods make use of thermodynamic and kinetic effects to reduce probe mismatches and failure of complementary probes to bind to DNA and RNA templates. Mapping and sequencing of the probed DNA and RNA samples are contemplated herein.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of priority under 35 U.S.C. §119(e)to U.S. Provisional Patent Application Ser. No. 61/754,258 filed Jan.18, 2013, which is hereby incorporated by reference in its entirety.

FIELD OF INVENTION

The present invention relates generally to methods for enhancing thebinding of oligonucleotide probes to DNA and RNA samples for analysis.Mapping and sequencing of the DNA and RNA samples are contemplatedherein.

BACKGROUND

A number of different approaches for sequencing nucleic acids exist. Thetraditional methods are the dideoxy-chain termination method describedby Sanger et al., Proc Natl. Acad. Sci. USA, (1977) 74: 5463-67 and thechemical degradation method described by Maxam et al., Proc. Natl. Acad.Sci. USA, (1977) 74: 560-564. Of these two methods, the Sanger procedurehas been the most widely used. The original Sanger method relied onradioactive labeling of the reaction products and separation of thereaction products by slab gel electrophoresis.

Both the Sanger and Maxam methods are time- and labor-intensive. Thestart of the Human Genome Project was the impetus for the development ofimproved, automated systems to perform Sanger sequencing. As a result,detection of fluorescence has replaced autoradiography and capillaryelectrophoresis has replaced the ultrathin slab gels originally used toseparate reaction products. Automated sequencers have been developed andare capable of processing large numbers of samples without operatorintervention.

The completion of the Human Genome Project has refocused the need fornew technologies that are capable of rapidly and inexpensivelydetermining the sequence of human and other genomes. There is has beenmuch discussion in recent years about personalized medicine. The visionof personalized medicine involves every individual having his or hercomplete genome sequenced at high accuracy and using this information toguide clinical care, specifically for risk stratification of patientsand pharmacogenomics.

In recent years, a number of technological advances have been developedenabling a great reduction in the cost of sequencing and substantiallyincreasing the amount of sequence data produced. Most sequencing methodscurrently available utilize optical detection for the determination ofthe DNA sequence. The most prevalent sequencing methods are referred toas sequencing by synthesis (SBS).

SBS typically consists of the stepwise synthesis of a strand of DNA thatis complementary to a template sequence from the target genome to besequenced. The SBS methods can be divided into those that are performedin batch mode and those that are performed in real-time. The batch modeprocesses rely on the stepwise synthesis of the new DNA strand with thelimitation that the synthesis is only allowed to proceed for onenucleotide position, for one nucleotide type, or for the combination ofone nucleotide position and one nucleotide type. The incorporation ofthe nucleotide occurs in parallel for large numbers of templates.Detection is achieved using a variety of methods.

The batch mode processes utilizing a single nucleotide type are used byRoche for pyrosequencing with the 454 platform. The Roche technology(see, e.g., Margulies et al. (2005) Nature, 437:376-380; U.S. Pat. Nos.6,274,320; 6,258,568; 6,210,891) utilizes pyrosequencing. The methoddepends on several enzymes and cofactors to produce luminescence when anucleotide is incorporated. A single nucleotide species is introducedinto a large number of small reaction vessels each containing multiplecopies of a single template. The incorporation of the nucleotide isaccompanied by light emission. When the reaction has run to completion,the reagents are washed from the reaction volumes and a next nucleotideand its required reagents are washed into the reactions. Each templateis thus extended in an iterative fashion, one nucleotide at a time.Multiple incorporations of the same nucleotide require the quantitativedetermination of the amount of light emitted. Homopolymer tracts intemplates may be difficult to accurately sequence as the incrementalamount of light emitted for each subsequent position in the homopolymerbecomes small compared to the total amount emitted.

In other variations of the SBS method, platforms by Helicos (see, e.g.,Quake et al Proc. Nat. Acad. Sci. USA (2003) 100: 3960-3964; U.S. Pat.Nos. 6,818,395; 6,911,345; 7,297,518; 7,462,449 and 7,501,245), Illumina(see, e.g., Bennett et al. Pharmacogenomics (2005) 6:373-382), andIntelligent Bio-Systems (see, e.g., Ju et al. Proc. Nat. Acad. Sci. USA(2006) 103:19635-19640) allow only the incorporation of a singlenucleotide at each step. Template strands are attached to a solidsupport and a primer sequence is annealed. A polymerase is used toextend the primer to make a complement to the template. The nucleotidesare derivatized such that after the incorporation of a singlenucleotide, the growing strand is incapable of further extension. Thenucleotides are further derivatized to make them fluorescent. In theHelicos technology, the four nucleotides are labeled with the samefluorescent tag. This requires that each nucleotide type be addedseparately. In contrast, the Illumina and Intelligent Bio-Systemstechnologies utilize four different fluorescent tags so that a mixtureof all four derivatized nucleotides may be added at the same time. Forboth technologies, the incorporation of a nucleotide is accompanied bythe appearance of fluorescence in the growing strand. In the case ofIllumina, the wavelength of the fluorescence emission indicates theidentity of the newly incorporated nucleotide. In the Helicostechnology, only a single nucleotide type is added at each cycle. Thus,the appearance of fluorescence at a position on the solid supportindicates the incorporation of the added nucleotide for that template.Templates that do not incorporate the nucleotide present in the reactionremain dark.

Following the observation of any incorporated fluorescence, the blockinggroups and fluorescent tags are removed prior to the next cycle.Multiple cycles result in the acquisition of sequence data for manytemplates in a single run. The instrumentation typical for thesetechnologies is said to allow for the automated acquisition of sequenceinformation for hundreds of thousands to millions of templates inparallel.

SBS methods may also be performed in real-time. In particular,polymerase is used to incorporate fluorescently labeled nucleotides andthe fluorescence is observed during DNA strand synthesis. The fournucleotides are labeled with different fluorescent tags. The fluorescenttags are attached to the terminal phosphate of the nucleotidetriphosphate. During incorporation of the nucleotide into the growingstrand the fluorophore is released to solution and the growing strandremains non-fluorescent. The identity of the incorporated strand isdetermined while the nucleotide resides in the active site of the enzymeand before the cleaved diphosphate is released to bulk solution.

The fluorescence of the incorporated nucleotide typically is measured inthe presence of a background fluorescence from a much largerconcentration of unincorporated nucleotide. Pacific Biosciences (see,e.g., U.S. Pat. Nos. 7,170,050; 7,302,146; 7,315,019; 7,476,503; and7,476,504) identifies the incorporated nucleotide based on the residencetime in the polymerase active site. Fluorescence emission from theactive site for an appropriate time indicates incorporation and theemission wavelength determines the identity of the incorporatednucleotide. Polymerase is attached to the bottom of zero-modewaveguides. Zero-mode waveguides are reaction cells whose dimensionslimit the passage of light from the excitation sources. Thus, onlyfluorescent tags close to the bottom surface of the reaction volume areexcited.

Other recently developed methods to sequence DNA rely on hybridizationand ligation. Both the SOLiD and Complete Genomics technologies rely onthe combination of hybridization and ligation. The SOLiD system (LifeTechnologies) immobilizes short template strands via an adapter. Aprimer and a pool of labeled oligonucleotides containing two fixedpositions and six degenerate positions is hybridized to the template.The primer hybridizes to the adaptor. Each pool consists of 16,384different sequences. Four fluorescent dyes are used to label theoligonucleotides in a pool in a fashion that creates four subsets fromthe sixteen combinations at the two fixed positions. Thus, eachfluorescent tag is associated with four of the sixteen possiblecombinations. Following hybridization, a ligase is added and any probesin the pool that hybridized contiguously with the primer are ligated tothe primer. The fluorescence of the hybridized and ligated product isdetermined. The fluorescence defines which subset of sequenceshybridized to the template and ligated to the primer. The terminal threebases and the associated fluorescent tag are cleaved from the hybridizedand ligated oligonucleotide. Subsequent rounds of another round ofhybridization, ligation, and cleavage are performed. In this firstseries of reactions, each cycle identifies a subset for the pair ofnucleotides in the template that is 5 nucleotides downstream from subsetof pairs that were identified in the last cycle. After several cycles,the primer, and the oligonucleotides that have been ligated to it, iswashed off the template.

The entire procedure is repeated starting with a primer that is onenucleotide shorter than the original primer, then with primers that aretwo, three, and four nucleotides shorter than the original primer. Thesesubsequent rounds shift the frame of interrogation so that the basesthat make-up the template strand can be identified from the unionbetween the two subsets of reaction that overlapped at that position.

Complete Genomics technology utilizes a similar hybridization andligation method (see, e.g., US Patent Application Publication Nos.20080234136; 20090005252; 20090011943; and 20090176652). In the CompleteGenomics technology, a primer is hybridized to an adaptor that isattached to the end of the template. A series of pools ofoligonucleotides is constructed. In each pool, the nucleotide at asingle position is identified by using four-color fluorescence. Theremaining positions are degenerate. The first pool is hybridized to thetemplate. Oligonucleotides that hybridize adjacent to the primer aresubsequently ligated. After washing excess oligonucleotides away, thefluorescence of the ligated oligonucleotide identifies the nucleotide atthe defined position in that pool. The ligated primer andoligonucleotide are washed off the template and the process is repeatedwith the next pool of oligonucleotides that probe the next position downfrom the primer.

The SBS and hybridization-ligation methods generate short pieces orreads of DNA sequence. While the short reads can be used to re-sequencehuman genomes, they are not favorable for the de novo assembly of humangenomes. With the recent realization that human genomes contain largenumbers of inversions, translocations, duplications, and indels (e.g.,mutations that include both insertions, deletions, and the combinationthereof), the quality of human genome data from short reads is even moresuspect. Genetic rearrangements are even more prevalent in cancer.

While short read technology methods that incorporate paired-end readshave been proposed and the length of the sequence data from thesetechnologies has increased incrementally over the last two years, it isclear that longer read technologies are necessary for the accurateassembly of human genome data.

In addition to the undesirable nature of short reads, all of the DNAsequencing methods described above employ optical detection. Thethroughput of optical methods limits the ultimate performancecharacteristics of any of these sequencing technologies. Optical methodsare capable of identifying single molecules. However, the time requiredto observe and accurately identify events is typically too slow to meetthe need for higher throughput. While the current generation ofsequencing technologies has lowered the cost of sequencing by orders ofmagnitude in comparison to the methods used to sequence the first humangenomes, the methods remain too slow, costly, and inaccurate for routineanalysis of human genomes.

In methods employing oligonucleotide probes, it is recognized that probebinding is subject to both false negatives and false positives. In thecase of false negatives, not every region on the analyte that iscomplementary to a probe necessarily has a probe bound thereto at agiven temperature, T. Likewise, in the case of false positives, probesbind to regions of the analyte that are not identically complementary,i.e., regions where, for example, there may be a single base mismatch.In both of these instances, errors may be produced in the final map orsequence data.

SUMMARY

A need exists for efficient methods and devices capable of rapid andaccurate nucleic acid sequencing for de novo assembly of human genomes.It is desirable to have long read lengths and to use as little nucleicacid template as possible. Moreover, single-molecule optical detectionof DNA has limitations with respect to sensitivity and therefore speed.Thus, there remains a need for improved methods and devices for theanalysis of biopolymers, including methods and devices for mapping andsequencing such biopolymers. A need also exists for improved methods bywhich probes are bound to samples to be analyzed to thereby reduce theoccurrence of false positive and false negative probe binding.

The embodiments of the invention provide assay methods for preparinganalyte samples for mapping and sequencing using nanopore, microchannelor nanochannel analysis devices.

Embodiments of the present invention relate broadly to the recognitionand use of thermodynamic effects and kinetic effects to improve bindingof oligonucleotide probes to DNA and RNA sample analytes. These effectsmay be used to reduce both false negatives that result from probesfailing to bind at complementary sites on the analyte as well falsepositives resulting from probes binding at sites having complementarymismatches. Improvements in probe binding provide enhanced accuracy whenusing the probes to derive maps and sequences of the samples beinganalyzed.

More particularly, in one aspect, embodiments of the invention relate toa method for preparing a biomolecule analyte which includes the steps ofproviding a single-stranded DNA or RNA template, hybridizing a pluralityof identical, sequence-specific oligonucleotide probes to the template,conducting a base extension reaction from a 3′ end of a hybridizedprobe, terminating the base-extension reaction, and allowing additionalunhybridized probes from the plurality of probes to hybridize to thetemplate.

One or more of the following features may be included. The baseextension reaction may be allowed to produce a double-stranded retion onthe single-stranded template of a length approximating the resolution ofa detection apparatus. The base extension reaction and the terminationmay carried out simultaneously. Following termination of the baseextension reaction, the analyte may be maintained at a temperature for atime sufficient to melt probe mismatches, e.g., sufficient to meltsubstantially all probe mismatches. This process may be carried out oneor more times. The probes may be provided with tags, such as doublestranded DNA, gold beads, quantum dots, or fluorophores. A at least aportion of the template or probes may be provided with a proteincoating, e.g., RecA, T4 gene 32 protein, f1 gene V protein, humanreplication protein A, Pf3 single-stranded binding protein, adenovirusDNA binding protein, or E. coli single-stranded binding protein.

The single-stranded DNA or RNA template may include one or moresecondary structures. In such cases, the secondary structure may bedenatured following termination of any of the base extension reactions.In particular, in another aspect of the invention, a method forpreparing a biomolecule analyte includes providing a single-stranded DNAor RNA template comprising one or more secondary structures. A pluralityof identical, sequence-specific oligonucleotide probes is hybridized tothe template. A base extension reaction is conducted from a 3′ end of ahybridized probe, The base-extension reaction is terminated. Thetemplate is denatured to break at least a portion of said one or moresecondary structures. The base extension reaction, termination, anddenaturing steps are then repeated at least one additional time with adifferent plurality of identical, sequence-specific oligonucleotideprobes to prepare the biomolecule analyte.

One or more of the following features may be included. The denaturingstep may include heating. At least a portion of the probes may includetagged probes. At least a portion of the template or probes may becoated with a protein. The base extension reaction and the terminationmay be carried out simultaneously.

In some embodiments of the invention, two or more probes may be used. Inanother aspect, embodiments of the invention include a method forpreparing a biomolecule analyte, the method including providing asingle-stranded DNA or RNA template, providing a first plurality ofidentical, sequence-specific oligonucleotide probes having a firstmelting temperature, and a second plurality of identical,sequence-specific oligonucleotide probes having a second meltingtemperature, the first melting temperature being higher than the secondmelting temperature. The first plurality of probes has a differentsequence than the second plurality of probes. The probes from the firstplurality are hybridized to the template at a temperature approximatelyequal to or below the first melting temperature, and a firstbase-extension reaction is conducted from a 3′ end of a hybridized firstprobe. The first base extension-reaction is terminated and thenadditional unhybridized probes from the first plurality of probes areallowed to hybridize to the template. A second base-extension reactionis then conducted from a 3′ end of a hybridized probe from the firstplurality of probes and is terminated. Finally, probes from the secondplurality of identical, sequence-specific oligonucleotide probes arehybridized to the template at a temperature approximately equal to orbelow the second melting temperature.

One or more of the following features may be included. At least aportion of the probes may include tagged probes. At least a portion ofthe template or probes may be coated with a protein. The first baseextension reaction and its termination may be carried outsimultaneously. The second base extension reaction and its terminationmay be carried out simultaneously.

Optionally, a third base-extension reaction may be conducted to extendfrom a 3′ end of a hybridized probe from the second plurality of probes.This is followed by termination of the base extension reaction andallowing additional unhybridized probes from the second plurality ofprobes to hybridize to the template. The third base extension reactionand its termination may be carried out simultaneously.

Additional base extension reactions may be allowed as desired. Forexample, a fourth base-extension reaction may be conducted in the atleast one single-stranded region from a 3′ end of a hybridized probefrom the second plurality of probes, and then terminated. The fourthbase-extension reaction and its termination may be carried outsimultaneously.

In some embodiments of the invention, an enzymatic ligation may besubstituted for the base extension reaction. Accordingly, in yet anotheraspect, embodiments of the invention include a method for preparing abiomolecule analyte by providing a single-stranded DNA or RNA template.A first plurality of identical, sequence-specific oligonucleotide probesand a second plurality of identical oligonucleotide probes are provided,The first plurality of probes and the second plurality of probes arehybridized to the template. An enzymatic ligation reaction is conductedto ligate hybridized probes to an adjacent probe from the secondplurality of identical oligonucleotide probes, terminating the ligationreaction, and allowing additional unhybridized probes from the firstplurality of probes to hybridize to the template.

One or more of the following features may be included. The probes fromthe second plurality may each include one degenerate or universal site.At least a portion of the first plurality of probes and/or secondplurality of probes may include tagged probes. At least a portion of thetemplate and/or first plurality of probes and/or second plurality ofprobes may be coated with a protein.

The biomolecule analytes prepared by the methods described herein may beused to map or sequence biomolecules using nanopores or fluidic channelssuch as nanochannels and microchannels. For example, any of thebiomolecule analytes prepared by the disclosed methods may be analyzedas follows. An apparatus may be provided, the apparatus having a firstfluid chamber, a second fluid chamber, a membrane positioned between thefirst and second chambers and a nanopore extending through the membranesuch that the first and second chambers are in fluid communication viathe nanopore. The biomolecule analyte may be introduced into the firstchamber and translocated from the first chamber through the nanopore andinto the second chamber. Changes in an electrical property across thenanopore may be monitored as the biomolecule analyte is translocatedtherethrough, the changes in the electrical property corresponding tolocations along the biomolecule analyte containing probes. The changesin the electrical property as a function of time may be recorded.

Moreover, any of the biomolecule analytes prepared by the disclosedmethods may be analyzed as follows. The biomolecule analyte may bedisposed in a fluidic nanochannel or microchannel. A potential may beapplied along the fluidic channel. The biomolecule analyte may betranslocated from a first end of the fluidic channel to a second end ofthe fluidic channel. Electrical properties may be detected as thebiomolecule analyte moves through the fluidic channel, the electricalproperties corresponding to at least one detector volume in the fluidicchannel, each detector volume being defined by two or more sensingelectrodes disposed along the length of the fluidic channel, with thedetected electrical signals indicating locations of hybridized probesalong the biomolecule analyte.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic depiction of a DNA molecule.

FIG. 2 is a schematic depiction of an RNA molecule.

FIG. 3 is a schematic depiction of a hybridizing oligonucleotide probe.

FIG. 4 is a schematic depiction of a single-stranded DNA moleculehybridized with two identical probes.

FIG. 5 is a schematic representation of a melting curve showing therelative amounts of double-stranded DNA versus single-stranded DNA overa range of temperatures.

FIGS. 6 a-6 d are schematic depictions of an assay preparation method inaccordance with an embodiment of the invention in which oligonucleotideprobes are hybridized to a single-stranded DNA or RNA template, a baseextension reaction is carried out, and a subsequent hybridization ofremaining unbound probes is allowed to proceed.

FIGS. 7 a and 7 b are schematic depictions of tagged probes useful inconnection with embodiments of the present invention.

FIGS. 8 a-8 d are schematic depictions of an assay preparation method inaccordance with an embodiment of the invention in which taggedoligonucleotide probes are hybridized to a single-stranded DNA or RNAtemplate, a base extension reaction is carried out, and a subsequenthybridization of remaining unbound tagged probes is allowed to proceed.

FIGS. 9 a-9 g are schematic depictions of an assay preparation method inaccordance with an embodiment of the invention in which two differentoligonucleotide probes are employed. In this embodiment, a first probeset is hybridized to a single-stranded DNA or RNA template using themethods of embodiments of the invention, and then a second probe set ishybridized to the analyte.

FIGS. 10 a-10 d are schematic depictions of an assay preparation methodin accordance with an embodiment of the invention in which ligation isused to enhance hybridization of probes to an analyte.

FIG. 11 a is a schematic depiction of an assay method in accordance withan embodiment of the invention showing a DNA molecule having a taggedprobe in a nanopore apparatus.

FIG. 11 b is a schematic depiction of an assay method in accordance withan embodiment of the invention showing a current measurement waveform asa DNA molecule having a tagged probe translocates through the nanoporeapparatus of FIG. 11 a.

FIG. 12 is a schematic depiction of an assay method in accordance withan embodiment of the invention showing a nanochannel or microchannelapparatus useful for mapping the analytes of embodiments of the presentinvention.

FIG. 13 a is a schematic depiction of an assay method in accordance withan embodiment of the invention showing an electrical potentialmeasurement as a DNA molecule having a tagged probe enters a detectionvolume in the apparatus of FIG. 12.

FIG. 13 b is a schematic depiction of an assay method in accordance withan embodiment of the invention showing an electrical potentialmeasurement as a tagged probe on a DNA molecule enters a detectionvolume in the apparatus of FIG. 12.

FIG. 13 c is a schematic depiction of an assay method in accordance withan embodiment of the invention showing an electrical potentialmeasurement as a tagged probe on a DNA molecule exits a detection volumein the apparatus of FIG. 12.

FIG. 13 d is a schematic depiction of an assay method in accordance withan embodiment of the invention showing an electrical potentialmeasurement as a DNA molecule having a tagged probe exits a detectionvolume in the apparatus of FIG. 12.

FIG. 14 is a schematic depiction of an assay method in accordance withan embodiment of the invention showing a nanochannel or microchannelapparatus having multiple detection volumes.

DETAILED DESCRIPTION

Embodiments of the present relate generally to methods for enhancing thebinding of oligonucleotide probes to DNA and RNA samples for analysis.

As used in this description and the accompanying claims, the followingterms shall have the meanings given, unless the context indicatesotherwise:

A “template” or “target” means a biomolecule, for example, havingsequence information that is to be determined using embodiments of thepresent invention. The target or template may be a biomolecule such asdeoxyribonucleic acid, a ribonucleic acid, a protein, or a polypeptide.The target or template may be single-stranded or double-stranded.

A “probe” means any molecule or assembly of molecules capable ofsequence-specific covalent or non-covalent binding to a template.Accordingly, a sequence-specific probe is capable of binding to aportion of the template having a complementary sequence.

A “biomolecule analyte” is any molecule or assembly of molecules, e.g.,a template having probes bound thereto, that is to be analyzed. Anexemplary biomolecule analyte may include a single-stranded DNA or RNAtemplate, with one or more sequence-specific oligonucleotide probeshybridized to a corresponding complementary portion of the template; abinding moiety may coat at least a portion of the single-stranded DNA orRNA template and/or probes.

A “tag” means a moiety that is attached to a probe in order to make theprobe more visible to a detector. These tags may be proteins,double-stranded DNA, single-stranded DNA, dendrimers, particles, orother molecules.

A “false negative” means that not every region on the analyte that iscomplementary to a probe necessarily has a probe bound thereto at agiven temperature, T.

A “false positive” means a probe that has bound to a region of theanalyte that is not identically complementary, i.e., a region where, forexample, there may be a single base mismatch.

In one embodiment, a biomolecule of interest is hybridized with theentire library of probes of a given length. For example, the biomoleculeof interest can be hybridized with the entire universe of 4096 (i.e.,4⁶) possible six-mers. The hybridization can be done sequentially (i.e.,one probe after another) or in parallel (i.e., a plurality ofbiomolecules of interest are each separately hybridized simultaneouslywith each of the possible probes.) Alternatively, the probes can beseparated from each other in both space and time. Additionally, morethan one probe type may be hybridized to the same biomolecule ofinterest at the same time.

The set of probes used to perform sequencing may be a subset of thecomplete library of probes of a given length, such as about 85%, 75%,65%, 55%, 45%, or 33% of the library. For instance, if sequencing isperformed on a biomolecule that starts as double-stranded DNA, then onlyone-half of the probes that make up a library may be needed. Othersubsets of the library may be designed to allow sequencing as well. Ifsome information concerning the target sequence is known prior toperforming the sequencing reaction, it may be possible to use a smallsubset of the total library. For instance, if the sequencing reaction isbeing performed to determine if single nucleotide polymorphisms arepresent with respect to a reference sequence, then a small number ofprobes with respect to the complete library may be used. Alternatively,the set of probes may not all be the same length. In an embodiment, aset of at least two probes may be used for hybridization, rather than anentire library of probes or subset thereof. In another embodiment,probes may be separated by (GC) content or other determinants of probebinding strength, in order to allow for optimization of reactionconditions. By separating the probes based on relative properties,multiple probes may be incorporated into a single hybridizationreaction. Further, the probes may be grouped based on their relatedoptimum reaction environment preferences. In yet another embodiment,pools of probes may be simultaneously hybridized to a biomolecule ofinterest. A pool of probes is a group of probes of differentcomposition, each of which may likely be present in many copies. Thecomposition of the probes may be chosen so as to reduce the chance ofcompetitive binding to the biomolecule of interest. Alternatively, thecomposition of multiple pools may be chosen so that the same competitivebinding is not present in all pools occupied by a single probe.

It should be understood that the methods of embodiments of the presentinvention are not intended to be limited solely to sequencing. As such,embodiments of the invention can be used to provide accurate maps ofanalytes. In particular, rather than employing a library of probes asdescribed above, in mapping applications, one or more sets ofsequence-specific probes can be used to map, with high accuracy, thespecific location of regions on the analyte which are complementary tosuch probes.

In still another embodiment, the probes may include tags, therebyenhancing detection as the hybridized probes translocate through thesequencing system. In addition, different tags may be used to helpdistinguish among the different probes. These tags may be proteins,double-stranded DNA, single-stranded DNA, particles, or other molecules.

It should be understood that embodiments of the invention are notintended to be limited strictly to DNA and RNA oligonucleotide probes.Rather, it is envisioned that oligonucleotide analog probes such asthose comprising LNAs, PNAs, 2′-methoxy nucleotide analogs, or otheranalogs may be used as well.

In one embodiment, the process of sequencing a biomolecule such assingle strands of DNA or RNA using one or more probes may performed asfollows. Suitable processes are also described in U.S. Ser. No.11/538,189, published as U.S. Publication No. 2007/0190542, incorporatedby reference herein in its entirety. Referring to FIG. 1, a DNA molecule1 is schematically depicted and is structured in two strands 2, 4positioned in anti-parallel relation to one another. Each of the twoopposing strands 2, 4 may be sequentially formed from repeating groupsof nucleotides 6 where each nucleotide 6 consists of a phosphate group,2-deoxyribose sugar and one of four nitrogen-containing bases. Thenitrogen-containing bases include cytosine (C), adenine (A), guanine (G)and thymine (T). DNA strands 2, 4 are read in a particular direction,from the top (called the 5′ or “five prime” end) to the bottom (calledthe 3′ or “three prime” end). Similarly, RNA molecules 8, asschematically depicted in FIG. 2, are polynucleotide chains, whichdiffer from those of DNA 1 by having ribose sugar instead of deoxyriboseand uracil bases (U) instead of thymine bases (T).

Traditionally, in determining the particular arrangement of the bases 6and thereby the sequences of the molecules, a process calledhybridization may be utilized. The hybridization process is theassociation, or binding, of two genetic sequences with one another. Thisprocess is predictable because the bases 6 in the molecules do not sharean equal affinity for one another. T (or U) bases favor binding with Abases while C bases favor binding with G bases. Binding is mediated viahydrogen bonds that exist between the opposing base pairs. For example,A binds to T (or U) using two hydrogen bonds, while C binds to G usingthree hydrogen bonds.

A hybridizing oligonucleotide, i.e., a probe, may be used to determineand identify the sequence of bases in the molecule of interest. FIG. 3illustrates a probe 10 that is a short DNA sequence having a knowncomposition. Probes 10 may be of any length depending on the number ofbases 12 that they include. For example, a probe 10 that includes sixbases 12 is referred to as a six-mer probe wherein each of the six bases12 in the probe 10 may be any one of the known four natural base typesA, T(U), C or G. Alternately, the probe may include non-natural bases.

In this regard, the total number of unique probes 10 in a library isdependent upon the number of bases 12 contained within each probe 10 andthe number of different types of bases in the probes. If only the fournatural bases are used in probe 10, the total number of probes in thelibrary is determined by the formula 4^(n) (four raised to the n power)where n is equal to the total number of bases 12 in each probe 10.Formulas for other arrangements or types of bases are well known in theart. Accordingly, the size of the probe library can be expressed as4^(n)-mer probes 10. For the purpose of illustration, in the context ofa six-mer probe, the total number of possible unique, identifiable probecombinations includes 4⁶ (four raised to the sixth power) or 4096 uniquesix-mer probes 10. The inclusion of non-natural bases allows for thecreation of probes that have spaces or wildcards therein in a mannerthat expands the versatility of the library, while reducing the numberof probes that may be needed to reach the final sequence result. Probesthat include universal bases organized into patterns with natural basesmay also be used, for example those described in U.S. Pat. Nos.7,071,324, 7,034,143, and 6,689,563, which are incorporated herein byreference in their entireties.

The process of hybridization using probes 10, as depicted in FIG. 4, maybegin by denaturing a double-stranded biomolecule, or by starting with asingle-stranded biomolecule. Denaturing is accomplished usually throughthe application of heat or chemicals, such that the hydrogen bondsbetween adjacent strands of the biomolecule are broken. The term“melting” may be used interchangeably with the term “denaturing”.

The hydrogen bonds between the two halves of an original double-strandedDNA may be broken, leaving two single strands of DNA whose bases are nowavailable for hydrogen bonding. After the biomolecule 14 has beendenatured, a single-stranded probe 10 may be introduced to thebiomolecule 14 to locate portions of the biomolecule 14 that have a basesequence that correlates in a complementary manner to the sequence thatis found in the probe 10. In order to hybridize the biomolecule 14 withthe probe 10, the denatured biomolecule 14 and a plurality of the probes10 having a known sequence are both introduced into a solution. Thesolution may be an ionic solution, such as a salt-containing solution.The mixture may be mixed to facilitate binding of the probes 10 to thebiomolecule 14 strand along portions thereof that have a matchedcomplementary sequence. Hybridization of the biomolecule 14 using theprobe 10 may be accomplished before the biomolecule 14 is introducedinto a nanopore sequencing apparatus or after the denatured biomolecule14 has been placed into the cis chamber of such an apparatus. In thiscase, after the denatured biomolecule has been added to the cis chamber,buffer solution containing probes 10 with a known sequence is also addedto the cis chamber and allowed to hybridize with the biomolecule 14before the hybridized biomolecule is translocated.

Probes are typically relatively short, e.g., 4-8 bases, and bind in afully complementary manner to templates. Nevertheless, in methodsemploying oligonucleotide probes, it is recognized that probe binding issubject to both false negative and false positives. In the case of falsenegatives, not every region on the analyte that is complementary to aprobe necessarily has a probe bound thereto at a given temperature, T.Likewise, in the case of false positives, probes occasionally bind toregions of the analyte that are not identically complementary, i.e.,regions where, for example, there may be a single base or multiple basemismatch. In each of these instances, errors may be produced in thefinal map or sequence data.

Embodiments of the present invention are based upon the recognition thatboth thermodynamic effects and kinetic effects may be used to enhanceprobe binding to an analyte and to reduce false negatives and falsepositives. For example, false positives may be reduced by inducingprobes bound with one or more base mismatches to become unbound by,e.g., controlling the temperature of the reaction. A melting curve forDNA is depicted schematically in FIG. 5. In that Figure, it can be seenthat at a temperature T₁ double-stranded DNA (dsDNA) remains in itsdouble-stranded configuration. At a higher temperature, T₂, the strandhas become completely denatured into two single-stranded DNA (ssDNA)templates. As applied to the binding of probes to ssDNA, this means thatas reaction temperature increases, probe binding decreases. At areaction temperature T_(M) (the melting temperature) approximately halfof all probes that can bind to the denatured analyte strand have doneso.

Thus, as shown in FIG. 5, when the probing reaction is maintained at T₁,there is approximately a 100% chance that a probe will be bound at acomplementary site. Likewise, at T₂, there is approximately a 0% chanceof a bound probe. At T_(M), there is approximately a 50% chance that aprobe will be bound. The process of probe binding is dynamic. As such,this does not mean that a particular subset of the complementary siteson the analyte remain bound with probes and the others remain unbound;rather, because probes continuously become bound and unbound over time,it means that at any given complementary recognition site on theanalyte, there is approximately a 50% chance that a probe will be boundat any given time. As a result of the foregoing, if one were to conducta probing reaction at T_(M), and then to run the resulting analyte in asequencing apparatus, only about 50% of the possible sites complementaryto the probe would be detected on each molecule, i.e., there would beapproximately 50% false negatives on each molecule. However, eachmolecule would have a different collection of complementary sites bound.If a sufficient number of molecules are detected all of thecomplementary binding sites may be determined. However, it is desirableto reduce false negatives so that each molecule is bound at a largeproportion of the complementary sites so that the identity of eachmolecule in a complex mixture may be determined with high accuracy.

As will be described in detail below, false negatives may be reducedthrough the use of a base extension reaction, such as a primer extensionreaction, utilizing for example, a polymerase and one or morenucleotides. In such reactions, which form a nucleic acid complementaryto a nucleic acid template, a primer complementary to a single-strandedDNA template is typically employed. Starting at the primer, a DNApolymerase may be used to add mononucleotides complementary to thetemplate at the 3′ end of the primer. Various base extension reactionswill be familiar to those of ordinary skill in the art. Note that if thetemplate comprises RNA, an RNA dependent DNA polymerase is employed.

One embodiment of the present invention relates to improved methods forthe preparation of biomolecule analytes. In the embodiment, shown inFIGS. 6 a-6 d, a denatured biomolecule analyte 15 is formed from asingle-stranded DNA (ssDNA) or RNA template 20 exposed to probes 10. Theprobes may be ssDNA, RNA or other modified nucleotides that selectivelyhybridize to the analyte. The template 20 is shown to include threeregions 25, referred to herein as probe recognition sites, which arecomplementary to the probes 10 being used. As such, each of the regions25 is a potential binding site for a probe 10. In this example, eachprobe 10 is a short, known ssDNA sequence. The probes 10 may be of anylength depending on the number of bases that they include. Each of theprobes is preferably of an identical sequence, thereby causing theprobes to selectively hybridize only to probe recognition sites 25 ofthe biomolecule template 20 that have a complementary sequence. Thetemplate 20 and probes 10 are depicted prior to hybridization in FIG. 6a. For purposes of clarity in FIGS. 6 a-6 d, probes 10 are shown havinga small dot at the 3′ end. This dot is not intended to signify aphysical structure; rather, it is included simply to designate the 3′end of the probe.

The biomolecule analyte 15 is shown in FIG. 6 b following hybridizationof probes to the biomolecule template. Note that as shown in FIG. 6 b,two probes (designated 10′) have become bound at two probe recognitionsites (designated 25′), while one probe 10 and probe recognition site 25remain unbound. Were one to analyze the analyte following this step, theunbound probe recognition site 25 would be read as a false negative.

Following hybridization, a base extension reaction, such as a primerextension reaction, utilizing for example, a polymerase and one or morenucleotides, is performed as depicted in FIG. 6 c. In such reactions,which form a nucleic acid complementary to a nucleic acid template, aprimer complementary to a single-stranded DNA template is typicallyemployed. In the present embodiment, each of the bound probes 10′ may beused as a primer in the base extension reaction. The probes are extendedfrom their 3′ ends along the template 20 to create duplex regions 40.The base extension reaction causes the probes 10′ to become moresecurely hybridized to the template.

It is preferred that the base extension reaction be limited in scope. Ifallowed to continue over extended lengths, the base extension mayoverwrite unbound probe recognition sites 25, rendering them aspermanent false negatives. Instead, rather than extending a longdistance from the 3′ end of each probe, the base extension reaction maybe terminated once the extensions have reached a length approximatingthe detection limits of the sequencing apparatus, such that thedouble-stranded region on the single-stranded template may have a lengthapproximating the resolution of a detection apparatus. This leavesunbound probe recognition sites 25 unoccupied for subsequent probingreactions. Extension reactions may be terminated by the addition ofdideoxynucleotides or other chain terminating nucleotides, such as3′-amino-modified oligonucleotides, at a suitable time after thebeginning of the extension reaction. Alternatively, the chainterminating nucleotides may be included with the cognate nucleotides inthe extension reaction. Suitable adjustment of the concentrations ofcognate and terminating nucleotides may be used to limit the extent ofelongation during the extension reaction.

The extension of a subset of probes 10 to form duplex 40 irreversiblybinds the probes to the template 20 under the reaction conditions andremoves them from the equilibrium between probes and template. Followingthe base extension reaction, hybridization of remaining unbound probes10, to unbound probe recognition sites 25, is allowed to proceed asdepicted in FIG. 6 d. Thus, over time, unbound probe recognition sites25 will subsequently become bound probe recognition sites 25′, therebyreducing the number of false negatives on the analyte. However, becausethe previously bound probes 10′ acted as primers for the base extensionreaction, they will have remained bound to the template. As such, itbecomes possible to have substantially all probe recognition sites boundby complementary probes. It should be understood that the steps depictedin FIGS. 6 a-6 d are intended as a schematic presentation and may havemultiple elements (e.g., extension and termination) occurringsimultaneously.

For example, in a typical biomolecule analyte preparation, thesingle-stranded template may be combined with a sequence-specificoligonucleotide probe, a polymerase, each of the four nucleotides usedto synthesize DNA, (deoxyadeninetriphosphate, dATP;deoxycytidinetriphosphate, dCTP; deoxyguanosinetriphosphate, dGTP; anddeoxy thymidinetriphosphate), as well as the dideoxy versions of each ofthose nucleotides (ddATP, ddCTP, ddGTP and ddTTP). Thus, when placed inthe presence of the template and maintained at the melting temperatureT_(M) of the probe, at any given time, approximately 50% of the probeswill be hybridized to the template. Likewise, if a temperature belowT_(M) of the probe is used, a higher percentage of probes will hybridizeto the template. This partial hybridization is depicted in FIG. 6 b.Among the hybridized probes, certain of them will act as primers for thepolymerase, and base extension will begin. The ratio of the deoxy- tothe dideoxy-forms of the nucleotide is used to regulate the length ofthe base extension, as the dideoxy-nucleotides terminate base extensionreactions. Thus, if the ratio of deoxy-nucleotides todideoxy-nucleotides is, for example, 100:1, it is expected that onaverage, base extensions will proceed for 100 bases prior totermination.

The extension reaction is preferably as short as possible, butsufficiently long to permanently anchor the probe to the template. Inpractice, an extension of 80 to 100 bases may be preferable. Moreover,preferably, the extension reaction should not extend for a distancelonger than can be resolved by a detector, e.g., currently about 300bases. The duration of the extension reaction, i.e., time beforetermination, depends on the polymerase used and the rate ofincorporation of nucleotides. Termination of extension may beaccomplised by removing polymerase, removing nucleotides, removingmagnesium (preferably with ethylenediaminetetraacetic acid (EDTA)) toinactivate the polymerase, heat killing the polymerase, or by usingmixtures of terminating and extending nucleotides.

The base extension is depicted in FIG. 6 c. Probes that have served asprimers for base extension reactions remain bound to the analyte. Over aperiod of time, additional unhybridized probes will become bound asshown in FIG. 6 d and act as primers for additional base extensionreactions. Thus, eventually most, if not all, probe recognition sitesbecome hybridized with complementary probes, and false negatives areeliminated. In some embodiments, it may be desirable to add the probesin a step prior to adding the polymerase and the deoxy- anddideoxy-nucleotides.

While the method described with reference to FIGS. 6 a-6 d is useful ineliminating false negatives, it should be understood that theelimination of false positives is a further enhancement of embodimentsof the present invention. False positives have previously been definedas instances where a probe has hybridized to a region of the analytethat is not identically complementary, i.e., a region where, forexample, there may be a single base or multi-base mismatch. As shown inFIG. 5, probes having mismatches generally have a lower T_(M) thanprobes having no mismatches.

The result of a lower T_(M) for probes having mismatches means that,during the base extension described with respect to FIGS. 6 a-6 d, theanalyte may be maintained at a temperature at or above the T_(M) for asufficient time, thereby causing mismatched probes to be denatured,i.e., melted, while extending correct hybridizations at complementarysites. By allowing the enzymatic extension to occur for longer times,false positives may be substantially reduced. The time and temperaturemay be sufficient to melt substantially all probe mismatches. In anotherembodiment of the invention, shown in FIGS. 8 a-8 d, the probes areprovided with tags that serve to make the probes more visible to adetector. Thus, as described above, suitable tags include proteins,double-stranded DNA, single-stranded DNA, dendrimers, particles, orother molecules.

Examples of two tagged probes are provided in FIGS. 7 a and 7 b. In FIG.7 a, a tagged probe 100 includes a probe 10 having a sequence and a tag60 connected to the 5′ end of the probe sequence 10 by a linker 50. Inthe embodiment shown in FIG. 7 a, the tag 60 may comprise a dsDNAsegment, however, any of a wide variety of chemical/biological tagsknown to those skilled in the art may be employed. In FIG. 7 b, a taggedprobe 110 includes a probe 10 having a sequence and a tag 70 connectedto the 5′ end of the probe sequence 10 by a linker 50. In the embodimentshown in FIG. 7 b, the tag 70 may comprise a gold bead, a quantum dot, afluorophore, etc. The tags make electrical fluctuations in sequencingsystems more noticeable as the hybridized probes translocate throughsuch systems. In addition, different tags may be used to helpdistinguish among different probes.

Thus, the embodiment shown in FIGS. 8 a-8 d, is identical to thatdepicted in FIGS. 6 a-6 d except that tagged probes are used.Specifically, a denatured biomolecule analyte 15 is formed from asingle-stranded DNA (ssDNA) or RNA template 20 exposed to tagged probes100. The probes may be ssDNA, RNA or other modified nucleotides thatselectively hybridize to the analyte. However, in this embodiment, theprobes include a tag as described above. As before, the template 20 hasbeen shown to include three probe recognition sites 25, which arecomplementary to the tagged probes 100 being used. The template 20 andtagged probes 100 are depicted prior to hybridization in FIG. 8 a.

The biomolecule analyte 15 is shown in FIG. 8 b following hybridizationof tagged probes to the biomolecule template. Two tagged probes(designated 100′) have become bound at two probe recognition sites(designated 25′), while one tagged probe 100 and probe recognition site25 remain unbound.

Following hybridization, a base extension reaction is performed asdepicted in FIG. 8 c. As before, each of the bound tagged probes 100′may be used as a primer in the base extension reaction. The probes areextended from their 3′ ends along the template 20 to create duplexregions 40. Because they are attached to the 5′ ends of the probes, thetags do not interfere with the base extension reaction.

Again, it is preferred that the base extension reaction be limited inscope. If allowed to continue over extended lengths, the base extensionmay overwrite unbound probe recognition sites 25, rendering them aspermanent false negatives. Instead, rather than extending a longdistance from the 3′ end of each probe, the base extension reaction maybe terminated once the extensions have reached a length approximatingthe detection limits of the sequencing apparatus. This leaves unboundprobe recognition sites 25 unoccupied for subsequent probing reactions.

Following the base extension reaction, hybridization of remainingunbound probes 100, to unbound probe recognition sites 25, is allowed toproceed as depicted in FIG. 8 d. As described previously, unbound proberecognition sites 25 will subsequently become bound probe recognitionsites 25′, thereby reducing the number of false negatives on theanalyte. However, because the previously bound probes 100′ acted asprimers for the base extension reaction, they will have remained boundto the template. As such, it becomes possible to have substantially allprobe recognition sites bound by complementary probes. As before, itshould be understood that the steps depicted in FIGS. 8 a-8 d areintended as a schematic presentation and may have multiple elementsoccurring (e.g., extension and termination) simultaneously.

As such, the analyte may be combined with a tagged sequence-specificoligonucleotide probe, a polymerase, each of the four nucleotides usedto synthesize DNA, and the dideoxy forms of each of those nucleotides.When placed in the presence of the analyte and maintained at T_(M) forthe tagged probe, at any given time, approximately 50% of the probeswill hybridize. As discussed previously, a higher percentage of probeswill hybridize if the hybridization reaction is carried out at atemperature below T_(M) for the tagged probe. Partial hybridization isdepicted in FIG. 8 b.

At least a portion of the tagged hybridized probes will act as primersfor the polymerase, and base extension will begin as depicted in FIG. 8c. Tagged probes that have served as primers for base extensionreactions remain bound to the analyte. Over a period of time, additionaltagged probes will become bound and act as primers for additional baseextension reactions as shown in FIG. 8 d. Over time most, if not all,probe recognition sites become hybridized with complementary taggedprobes, and false negatives are eliminated. As described previously, insome embodiments, it may be desirable to add the probes in a step priorto adding the polymerase and the deoxy- and dideoxy-nucleotides.

In a further embodiment of the invention, two or more pluralities ofprobes may be used. In the embodiment, shown in FIGS. 9 a-9 g, adenatured biomolecule analyte 15 is once again formed from asingle-stranded DNA (ssDNA) or RNA template 20 exposed to a plurality ofidentical, sequence-specific oligonucleotide probes, i.e., a set offirst probes 75 and a different plurality of identical,sequence-specific oligonucleotide probes, i.e., a set of second probes76. The probes may be ssDNA, RNA or other modified nucleotides thatselectively hybridize to the analyte. The probes of the first probe set75 are identical to one another, and the probes of the second probe set76 are also identical to one another, however the probes of the firstset 75 are different than those of the second set 76. First probes 75may have a melting temperature T_(M1) which is higher than the meltingtemperature T_(M2) of the second probes 76. The template 20 is shown toinclude two regions 25, referred to herein as first probe recognitionsites, which are complementary to first probes 75 and one region 26,referred to herein as a second probe recognition site, which iscomplementary to second probes 76. As such, each of the regions 25 and26 is a potential binding site for a probe.

The template 20 and first and second probes 75, 76 are depicted prior tohybridization in FIG. 9 a. As before, in FIGS. 9 a-9 g, probes are shownhaving a small dot at the 3′ end. This dot is not intended to signify aphysical structure; rather, it is included simply to designate the 3′end of the probe. Hybridization is preferably carried out at atemperature that is at or below T_(M1), but above T_(M2). Because T_(M1)is higher than T_(M2), the melting temperature of the second probe 76,the process conditions favor hybridization of the first probe 75.

The biomolecule analyte 15 is shown in FIG. 9 b once hybridization ofprobes to the biomolecule template has begun. Note that as shown in FIG.9 b, one first probe (designated 75′) has become bound at a first proberecognition site (designated 25′), while one first probe 75 and firstprobe recognition site 25 remain unbound. Second probe 76 and secondprobe recognition site 26 also remain unbound.

Following hybridization of the first probe, a base extension reactionoff of the 3′ end of first bound probe 75 is performed as depicted inFIG. 9 c. The base extension 40 causes bound first probes 75′ to becomemore securely hybridized to the template.

As before, it is preferred that the base extension reaction be limitedin scope to prevent the extensions from overwriting unbound first 25 andsecond 26 probe recognition sites. This leaves unbound probe recognitionsites unoccupied for subsequent probing reactions.

Following the base extension reaction, hybridization of remainingunbound first probes 75, to unbound first probe recognition sites 25, isallowed to proceed as depicted in FIG. 9 d. Thus, over time, unboundfirst probe recognition sites 25 will subsequently become bound firstprobe recognition sites 25′, thereby reducing the number of falsenegatives on the analyte. However, because the previously bound firstprobes 75′ acted as primers for the base extension reaction, they willhave remained bound to the template. As such, it becomes possible tohave substantially all first probe recognition sites bound bycomplementary probes. Furthermore, since the temperature of the reactionis preferably higher than the melting temperature T_(M2) of the secondprobes 76, binding of the first probes is favored over the secondprobes.

As additional first probes 75′ are hybridized to additional first probebinding sites 25′, base extension reactions from the newly hybridizedfirst probes 75′ take place as depicted in FIG. 9 e.

Once enough time has elapsed to allow substantially all first probes 75′to hybridize, the temperature is lowered to T_(M2) or below and secondprobes 76 are allowed to hybridize to their complementary second probebinding sites 26. This is shown in FIG. 9 f.

Following hybridization of the second probes 76′ at second proberecognition sites 26′, bound second probes 76′ act as primers for a baseextension reaction. This reaction, the result of which is depicted inFIG. 9 g, serves the same purpose as before; namely, to prevent boundsecond probes 76′ from melting from the second probe recognition site.Process conditions are maintained for a period of time sufficient toallow substantially all second probe recognition sites 26 to becomebound by second probes 76.

It should be understood that the steps depicted in FIGS. 9 a-9 g areintended as a schematic presentation and may have multiple elements(e.g., extension and termination) occurring simultaneously. Although notdepicted in FIGS. 9 a-9 g, it is to be further understood that either orboth of the first 75 and second 76 probe sets may include detectabletags.

In another embodiment of the invention, rather than using a baseextension reaction, a ligation reaction is carried out to secure probesto the analyte. The use of ligases to enhance probe binding is desirablein that ligases join probes with higher efficiency if the probes areperfectly complementary to the regions of the target analyte to whichthey are hybridized. As such, the use of ligases reduces enhancedbinding of probes that contain mismatches with the analyte.

As used herein, the term “ligation” refers to a method of joining two ormore nucleotides to one another. In general, the ligation methodsdescribed herein utilize enzymatic ligation using ligases. Such ligasesinclude, but are not limited to DNA ligase I, DNA ligase II, DNA ligaseIII, DNA ligase IV, E. coli DNA ligase, T4 DNA ligase, T4 RNA ligase 2,T4 RNA ligase 2, T7 ligase, T3 DNA ligase, and thermostable ligases,including without limitation, Taq ligase and the like.

The relevance of ligases to the methods of embodiments of the presentinvention is illustrated schematically in FIGS. 10 a-10 b. In FIG. 10 a,an 11-mer portion of the analyte 15 having the sequence TCAGAGCNNNA isshown. A 6-mer probe 10 having the sequence AGTCTC is shown hybridizedto its complementary probe recognition site 25, (i.e., the sequenceTCAGAG). A 5-mer oligonucleotide probe 11, optionally having degeneratesites (N) is hybridized to the analyte immediately adjacent to the 3′end of the probe 10. It is noted that probe 11 need not includedegenerate sites;

rather it could be perfectly complementary or it could include universalbases which hybridize equally well with each of the four cognate bases.Since the probe 10 is perfectly complementary to the probe recognitionsite 25, when an enzymatic ligation is carried out, the 5-mer probe 11becomes ligated to the probe 10, enhancing the ability of the probe toremain bound to the analyte even at temperatures above the meltingtemperature of the probe. (The ligation is represented in the Figure bya dot).

FIG. 10 b also shows a probe 10 and a 5-mer oligonucleotide probe 11hybridized to the analyte. Unlike FIG. 10 a however, a mismatch 13 ispresent between the probe 10 and the probe recognition site 25. As aresult, upon carrying out an enzymatic ligation reaction, probes 10 and11 do not become ligated. Thus, the probe 10 receives no enhancement ofits bond to the analyte and both probe 10 and the 5-mer probe 11 may bemelted from the analyte. Consequently, it is seen that the use of theligation reaction enhances the accuracy of the probes by enhancingbonding only of those probes that are perfectly matched to theircorresponding probe recognition sites.

The use of the ligation reaction as applied to embodiments of thepresent invention is shown in FIGS. 10 c and 10 d. In FIG. 10 c, theanalyte 15 includes three probe recognition sites 25. Each of these isshown with a hybridized probe. Two of the probes 10′ are perfectlycomplementary to their corresponding probe recognition sites, but oneprobe 10″ includes a mismatch. Several 5-mer probes 11, of the typedescribed with respect to FIG. 10 a are hybridized as well. One of theprobes 11′ is directly adjacent to the 3′ end of a perfectly hybridizedprobe 10′, and one of the probes 11″ is directly adjacent to the 3′ endof a hybridized probe 10″ having a mismatch.

Upon conducting an enzymatic ligation reaction, only the probe 10′ thatis perfectly complementary to its corresponding probe recognition sitebecomes ligated to the adjacent 5-mer probe 11′. Although 5-mer probe11″ is directly adjacent to the 3′ end of bound probe 10″, the existenceof a mismatch in the probe 10″ binding prevents probe 11″ from ligatingto probe 10″. Thus, as shown in FIG. 10 d, upon heating only the ligatedprobe 10′ and probe 11′ remain hybridized to the analyte. The mismatchedprobe 10″ and its adjacent 5-mer probe 11″ as well as other non-adjacentprobes 10 and 11 are readily melted from the analyte.

The steps depicted in FIGS. 10 a-10 d are intended as a schematicpresentation and may have multiple elements occurring simultaneously. Itshould be further understood that the description of probe 10 as a 6-meroligonucleotide, and of probe 11 as a 5-mer oligonucleotide, areintended as examples for illustration purposes only. Probes may be ofany length having utility in the applications described. Although notdepicted in FIGS. 10 a-10 d, it is to be further understood that probesmay include detectable tags.

In one embodiment of the invention, the incidence of false negativeevents resulting from secondary structure in the ssDNA or RNA templateis reduced. Specifically, one reason for the inability of a probe tobind to a single-stranded DNA template, is the formation of secondarystructure in that DNA template. A secondary structure is formed when asingle-stranded molecule hybridizes to itself to form a hairpin, loop,etc. Secondary structures are generally undesirable in the methodsdisclosed herein, as they may appear to a detector as a hybridizedprobe. In addition, secondary structures may compete with the binding ofthe probe to a complementary site. Finally, secondary structures maypromote clogging during translocation of templates or biomoleculeanalytes. The amount of false negative binding due to the secondarystructure is determined by the relative stability of the probe boundstructure as compared to that of the secondary structure. Thus, if thesecondary structure has weak binding when the probe is tightly bound,very few false negative events are expected. However, if the secondarystructure is very stable at the T_(M) of the probe, then a high numberof false negative binding events are expected because only a smallproportion of the template are available for binding by probes.

It is preferred that the template DNA of the biomolecule analyte behybridized with the probe under conditions such that some of thecomplementary sites involved in the formation of secondary structure areopen. That is, conditions may be chosen to insure that the equilibriumof the secondary structure does not result in 100% of the template beingin the form of the secondary structure. As such, portions of the ssDNAtemplate that do not have secondary structure are available for bindingby the probe. Thus, if the hybridization is conducted at the T_(M) ofthe secondary structure, at any instant, 50% of the molecules have nosecondary and are available for binding by the probe. The conditions maybe selected such that the template DNA available for binding by theprobe become completely bound or such that only a fraction of theavailable binding sites may be bound.

Structures in which the probe is hybridized to the template may beextended by a polymerase. As described above, it may be desirable toperform a limited extension of the probe. Following extension of boundprobe, the template is heated in order to melt the secondary structure.The template may then be re-hybridized with the excess probe at theT_(M) of the secondary structure. As before, the remainingsingle-stranded template is 50% available for binding by the probe. Thiscycle of hybridization, extension of probe, and denaturation may berepeated as many times as necessary to reduce the false negative rateresulting from the secondary structure. Temperatures or conditions otherthan the T_(M) of the secondary structure may be used to perform thesame conversions. Further, the conditions may be changed during eachcycle of hybridization, extension, and denaturation.

In a further embodiment of the invention, the analyte, i.e., at least aportion of the template or probes, may be coated to enhance its abilityto be detected. Coating methods are described in detail in co-pending USPatent Application Publication No. 20100243449, the teachings of whichare incorporated by reference. Broadly, coated biomolecules typicallyhave greater uniformity in their translocation rates, which leads to adecrease in positional error and thus more accurate sequencing. Due toits increased diameter, a coated biomolecule generally translocatesthrough a sequencing system at a slower speed than a non-coatedbiomolecule. The translocation is preferably slow enough so that asignal can be detected during its passage from a first chamber into asecond chamber. Exemplary binding moieties include proteins such as, forexample, RecA, T4 gene 32 protein, f1 geneV protein, human replicationprotein A, Pf3 single-stranded binding protein, adenovirus DNA bindingprotein, and E. coli single-stranded binding protein.

The translocation rate or frequency may be further regulated byintroducing a salt gradient between the chambers. Exemplary saltconcentration ratios of the cis to the trans side of the chamber mayinclude, but are not limited to, 1:2, 1:4, 1:6, and 1:8. For example,salt concentrations may range from about 0.5 M KCl to about 1M KCl onthe cis side and from about 1M KCl to about 4M KCl on the trans side.The signal is preferably strong enough to be detected using knownmethods or methods described herein. Exemplary signal-to-noise ratiosinclude, but are not limited to, 2:1, 5:1, 10:1, 15:1, 20:1, 50:1,100:1, and 200:1. With a higher signal-to-noise ratio, a lower voltagemay be used to effect translocation.

The analytes described herein may be configured for detection ofpositional information in a nanopore and/or a fluidic channel, i.e., amicrochannel or nanochannel system. Mapping of analytes may be carriedout using electrical detection methods employing nanopores, nanochannelsor microchannels using the methods described in U.S. patent applicationSer. No. 12/789,817, filed May 28, 2010, the teachings of which havepreviously been incorporated herein by reference. It is contemplatedthat such methods may be applied to analytes having either or bothtagged and untagged probes.

In one embodiment, current across a nanopore is measured duringtranslocation of a DNA strand through the nanopore as shown in FIG. 11a. When used in embodiments of the present invention, a nanopore mayhave a diameter selected from a range of about 1 nm to about 1000 nm.More preferably the nanopore has a diameter that is between about 2.3 nmand about 100 nm. Even more preferably the nanopore has a diameter thatis between about 2.3 nm and about 50 nm. Changes in an electricalproperty across a nanopore may be monitored as the analyte istranslocated therethrough, with changes in the electrical property beingused to distinguish regions of the analyte including probes, and regionsof the analyte lacking probes.

Specifically, for nanopore 105, a measurable current 115 produced byelectrodes 120, 122 runs parallel to the movement of the target analyte15, i.e., a DNA molecule having a tagged probe 100′. Variations incurrent are a result of the relative diameter of the target analyte 15as it passes through the nanopore 105. This relative increase in volumeof the target analyte 15 passing through the nanopore 105 causes atemporary interruption or decrease in the current flow through thenanopore, resulting in a measurable current variation. Portions of thetarget analyte 15 including a tagged probe 100′ are larger in diameterthan portions of the target analyte that do not include a probe. As aresult, when the tagged probe 100′ passes through the nanopore 105,further interruptions or decreases in the current flow betweenelectrodes 120, 122 occurs. These changes in current flow are depictedin the waveform 200 in FIG. 11 b.

Analysis of the waveform 200 permits differentiation between regions ofthe analyte including probes and regions without probes, based, at leastin part, on the detected changes in the electrical property, to therebydetermine probe locations and map at least a portion of thedouble-stranded DNA template. In FIG. 11 b, the waveform 200 depicts thechanges in a detected electrical property as the analyte passes throughthe nanopore, and may be interpreted as follows. Current measurement 210represents measured current prior to passage of the DNA molecule 15,i.e., the analyte, through the nanopore 105 from the cis side to thetrans side. As the analyte enters the nanopore 105, from the cis side ofthe nanopore, the current is partially interrupted forming a firsttrough 220 in the recorded current. Once the probe 100′ bound to theanalyte enters the nanopore 105, a further decrease in current occurs,causing a deeper, second trough 230 in the current measurement. Uponpassage of the probe 100′ entirely through the nanopore 105, a distalportion of the analyte may remain in the nanopore. This causes themeasured current 240 to rise to approximately the level of the firsttrough 220. Finally, once the entire analyte has passed completelythrough the nanopore 105 to the trans side, the measured current 250returns to a level approximating that of the initial level 210. Thecurrent variation measurements are recorded as a function of time.

As a result, the periodic variations in current indicate where, as afunction of relative or absolute position, the probes 100′ havehybridized to complementary regions on the analyte 15. Since the probesare bound at probe recognition sites for the specific sequences of theprobe, the relative or absolute position of the specific sequencesassociated with the recognition site for the particular probe employedmay be determined. This allows mapping of those specific sequences onthe analyte. Multiple maps produced using multiple probes may begenerated.

The use of a binding moiety, such as the protein RecA, may furtherenhance detection of analytes and probe regions on analytes because theadded bulk of the binding moiety coating causes greater currentdeflections.

In another embodiment, an electrical property such as electricalpotential or current is measured during translocation of a DNA strandthrough a nanochannel or microchannel as shown in FIGS. 12 through 14.One embodiment of a fluidic channel apparatus is shown schematically inFIG. 12. In FIG. 12, the apparatus 300 includes a fluidic microchannelor nanochannel 302. The fluidic channel may be a microchannel having awidth selected from a range of about 1 μm to about 25 μm or ananochannel having a width selected from a range of about 10 nm to about1000 nm. In the case of a microchannel, the depth may be selected from arange of about 200 nm to about 5 μm, whereas in the case of ananochannel, the depth may be selected from a range of about 10 nm toabout 1000 nm. In either case, the channel may have a length selectedfrom a range of about 1 μm to about 10 cm.

A first pair of electromotive electrodes 304, 304′ is connected to avoltage source 306 and positioned in a spaced apart relationship in thechannel. When a potential is applied to the electromotive electrodes,these electrodes provide an electrical current along the channel and maybe used to provide or enhance a driving force 308 to an analyte 15 inthe channel. Other driving forces such as pressure or chemical gradientsare contemplated as well. A second pair of electrodes 312, 312′, i.e.,detector electrodes, is positioned preferably substantiallyperpendicular to the channel in a spaced apart relationship to define adetection volume 314. The second pair of detector electrodes 312, 312′is connected to a detector 316, such as a voltmeter, which monitors anelectrical property in the detection volume 314. In an embodiment wherethe detector 316 is a voltmeter, an electrical potential between thepair of detector electrodes 312, 312′, is measured across the detectionvolume 314.

The operation of the device is depicted schematically in FIGS. 13 a-13 din which changes in an electrical property across a fluidic channel aremonitored, as the analyte 15 is translocated therethrough, with thechanges in the electrical property being indicative of probe-containingand probe-free regions. In FIGS. 13 a-13 d, the first pair ofelectromotive electrodes 304, 304′ and the current source 306 have beenomitted for clarity. In FIG. 13 a, the fluidic channel 302 contains ananalyte 15 traveling therethrough. An electrical property, in this caseelectrical potential, is measured and recorded across the detectionvolume 314 by the detector electrodes 312, 312′ and the detector 316.The analyte 15 is a DNA template upon which probes have been hybridizedusing the methods described previously. The DNA template and/or theprobe may be coated with a binding moiety, such as the protein RecA, toenhance detection.

Prior to the entry of the analyte 15 into the detection volume 314, asubstantially constant voltage 322 is measured across the detectionvolume. This voltage is shown in the waveform 320 of FIG. 13 a. As theanalyte 15 enters the detection volume 314, it causes an interruption ordecrease in the electrical property measured in the detection volume.This interruption or decrease causes a first trough 324 to be exhibitedin the waveform 320.

FIG. 13 b shows the device and waveform 320 once the portion of thetarget analyte 15 including the probe has entered the detection volume314. Entry of the probe into the detection volume 314 causes a furtherinterruption or decrease in the electrical property measured in thedetection volume. This further interruption or decrease causes a secondtrough 326 to be exhibited in the waveform 320.

In FIG. 13 c, the portion of the analyte 15 containing the probe hasexited the detection volume 314; however, a distal portion of theanalyte 15 may still be present in the detection volume. As a result,the waveform 320 has returned to a level 328 approximating that detectedwhen the initial portion of the analyte first entered the detectionvolume.

Finally, as shown in FIG. 13 d, the analyte 15 has fully exited thedetection volume 314. As a result, the waveform 320 has returned to alevel 330 approximating that detected prior to initial entry of theanalyte into the detection volume. Analysis of the waveform 320 permitsdifferentiation between probe-containing and probe-free regions of theanalyte, based, at least in part, on the detected changes in theelectrical property. As such, it is possible to determine probelocations and map at least a portion of the analyte.

Another embodiment of a fluidic channel apparatus is shown in FIG. 14.In FIG. 14, the apparatus 400 comprises a fluidic microchannel ornanochannel 402. As before, the fluidic channel may be a microchannelhaving a width selected from a range of about 1 μm to about 25 μm or ananochannel having a width selected from a range of about 10 nm to about1 μm. In the case of a microchannel, the depth may be selected from arange of about 200 nm to about 5 μm, whereas in the case of ananochannel, the depth may be selected from a range of about 10 nm toabout 1 μm. In either case, the channel may have a length selected froma range of about 1 μm to about 10 cm.

A first pair of electromotive electrodes 404, 404′ is connected to avoltage source 406 and positioned in a spaced apart relationship in thechannel. When a potential is applied to the electromotive electrodes,these electrodes provide an electrical current along the channel and maybe used to provide or enhance a driving force 408 to an analyte 15 inthe channel. Other driving forces such as pressure or chemical gradientsare contemplated as well. Multiple detector electrodes 412, 414, 416,418, are positioned preferably perpendicular to the channel in a spacedapart relationship to define a plurality of detection volumes betweenadjacent detector electrodes. Thus, as seen in FIG. 14, detectorelectrodes 412 and 414 define detection volume 420, detector electrodes414 and 416 define detection volume 422, and detector electrodes 416 and418 define detection volume 424. The detector electrodes are eachconnected to detectors 426, 428, 430 such as voltmeters, which monitoran electrical property in each detection volume. In the embodiment wherethe detectors are voltmeters, a drop in electrical potential is measuredacross each detection volume. Operation of the apparatus is similar tothat of the system of FIGS. 13 a-13 d, with the exception thatadditional waveforms are generated due to the presence of additionaldetection volumes. The additional waveforms may be combined to furtherimprove the quality of the data being generated by the device.

It should be understood that number of detector electrodes and detectionvolumes is not intended to limited to those depicted in FIG. 14. Rather,any number of detection volumes may be included along the length of thefluidic channel. Further, the detector electrodes and detection volumesneed not be evenly spaced, evenly sized or directly adjacent to oneanother. Various detection volume sizes, spacing and configurations arecontemplated.

EQUIVALENTS

Those skilled in the art will readily appreciate that all parameterslisted herein are meant to be exemplary and actual parameters dependupon the specific application for which the methods and materials ofembodiments of the present invention are used. It is, therefore, to beunderstood that the foregoing embodiments are presented by way ofexample only and that, within the scope of the appended claims andequivalents thereto, the invention may be practiced otherwise than asspecifically described.

The described embodiments of the invention are intended to be merelyexemplary and numerous variations and modifications will be apparent tothose skilled in the art. All such variations and modifications areintended to be within the scope of the present invention as defined inthe appended claims.

What is claimed is:
 1. A method for preparing a biomolecule analyte, themethod comprising: a. providing a single-stranded DNA or RNA template;b. hybridizing a plurality of identical, sequence-specificoligonucleotide probes to the template; c. conducting a base extensionreaction from a 3′ end of a hybridized probe; d. terminating thebase-extension reaction; and e. allowing additional unhybridized probesfrom the plurality of probes to hybridize to the template to prepare thebiomolecule analyte.
 2. The method of claim 1, wherein the baseextension reaction is allowed to produce a double-stranded region on thesingle-stranded template of a length approximating the resolution of adetection apparatus.
 3. The method of claim 2, wherein the baseextension reaction and the termination are carried out simultaneously.4. The method of claim 1, further comprising: following termination ofthe base extension reaction, maintaining the hybridized template under aset of conditions including temperature and for a time sufficient tomelt probe mismatches.
 5. The method of claim 4, wherein the time andtemperature are sufficient to melt substantially all probe mismatches.6. The method of claim 1, wherein at least a portion of the probescomprises tagged probes.
 7. The method of claim 6, wherein the tags onthe probes include at least one of double stranded DNA, gold beads,quantum dots, or fluorophores.
 8. The method of claim 1, wherein atleast a portion of the template or probes is coated with a protein. 9.The method of claim 8, wherein the protein includes at least one ofRecA, T4 gene 32 protein, f1 geneV protein, human replication protein A,Pf3 single-stranded binding protein, adenovirus DNA binding protein, orE. coli single-stranded binding protein.
 10. A method for preparing abiomolecule analyte, the method comprising: a. providing asingle-stranded DNA or RNA template comprising one or more secondarystructures; b. hybridizing a plurality of identical, sequence-specificoligonucleotide probes to the template; c. conducting a base extensionreaction from a 3′ end of a hybridized probe, d. terminating thebase-extension reaction; e. denaturing the template to break at least aportion of said one or more secondary structures; and f. repeating stepsb, c, d, and e at least one additional time with a different pluralityof identical, sequence-specific oligonucleotide probes to prepare thebiomolecule analyte.
 11. The method of claim 10, wherein the denaturingstep comprises heating.
 12. The method of claim 10, wherein at least aportion of the probes comprises tagged probes.
 13. The method of claim10, wherein at least a portion of the template or probes is coated witha protein.
 14. The method of claim 10, wherein the base extensionreaction and the termination are carried out simultaneously.
 15. Amethod for preparing a biomolecule analyte, the method comprising: a.providing a single-stranded DNA or RNA template; b. providing a firstplurality of identical, sequence-specific oligonucleotide probes havinga first melting temperature, and a second plurality of identical,sequence-specific oligonucleotide probes having a second meltingtemperature, the first melting temperature being higher than the secondmelting temperature and the first plurality of probes having a differentsequence than the second plurality of probes; c. hybridizing probes fromthe first plurality of identical, sequence-specific oligonucleotideprobes to the template at a temperature between the second meltingtemperature and the first melting temperature; d. conducting a firstbase extension reaction from a 3′ end of a hybridized probe from thefirst plurality of probes; e. terminating the first base-extensionreaction; f. allowing additional unhybridized probes from the firstplurality of probes to hybridize to the template; g. conducting a secondbase-extension reaction from a 3′ end of a hybridized probe from thefirst plurality of probes; h. terminating the second base-extensionreaction; and i. hybridizing probes from the second plurality ofidentical, sequence-specific oligonucleotide probes to the template at atemperature equal to or below the second melting temperature to preparethe biomolecule analyte.
 16. The method of claim 15, wherein at least aportion of the probes comprises tagged probes.
 17. The method of claim15, wherein at least a portion of the template or probes is coated witha protein.
 18. The method of claim 15, wherein the first base extensionreaction and its termination are carried out simultaneously.
 19. Themethod of claim 15, wherein the second base extension reaction and itstermination are carried out simultaneously
 20. The method of claim 15,further comprising, after probes from the second plurality of identical,sequence-specific oligonucleotide probes are hybridized to the analyte,carrying out the additional steps of: j. conducting a thirdbase-extension reaction in the at least one single-stranded region froma 3′ end of a hybridized probe from the second plurality of probes; k.terminating the third base-extension reaction; and allowing additionalunhybridized probes from the second plurality of probes to hybridize tothe template.
 21. The method of claim 20, wherein the third baseextension reaction and its termination are carried out simultaneously.22. The method of claim 20, further comprising the steps of: m.conducting a fourth base-extension reaction in the at least onesingle-stranded region from a 3′ end of a hybridized probe from thesecond plurality of probes, and n. terminating the fourth base-extensionreaction.
 23. The method of claim 22, wherein the fourth base extensionreaction and its termination are carried out simultaneously.
 24. Amethod for preparing a biomolecule analyte, the method comprising: a.providing a single-stranded DNA or RNA template; b. providing a firstplurality of identical, sequence-specific oligonucleotide probes and asecond plurality of identical oligonucleotide probes, the firstplurality being different from the second plurality; c. hybridizing thefirst plurality of probes and the second plurality of probes to thetemplate; d. conducting an enzymatic ligation reaction to ligatehybridized probes from the first plurality to adjacent probes from thesecond plurality; and e. allowing additional unhybridized probes fromthe first plurality of probes to hybridize to the template to preparethe biomolecule analyte.
 25. The method of claim 24 wherein the probesfrom the second plurality each include at least one degenerate oruniversal site.
 26. The method of claim 24, wherein at least a portionof the first plurality of probes comprises tagged probes.
 27. The methodof claim 24, wherein at least a portion of the second plurality ofprobes comprises tagged probes.
 28. The method of claim 24, wherein atleast a portion of the template is coated with a protein.
 29. The methodof claim 24, wherein at least a portion of the first plurality of probesis coated with a protein.
 30. The method of claim 24, wherein at least aportion of the second plurality of probes is coated with a protein. 31.A method for analyzing a biomolecule analyte comprising the steps of: a.preparing the biomolecule analyte using the method of claim 1; b.providing an apparatus having a first fluid chamber, a second fluidchamber, a membrane positioned between the first and second chambers anda nanopore extending through the membrane such that the first and secondchambers are in fluid communication via the nanopore; c. introducing thebiomolecule analyte into the first chamber; d. translocating thebiomolecule analyte from the first chamber through the nanopore and intothe second chamber; e. monitoring changes in an electrical propertyacross the nanopore as the biomolecule analyte is translocatedtherethrough, the changes in the electrical property corresponding tolocations along the biomolecule analyte containing probes; and f.recording the changes in the electrical property as a function of time.32. A method for analyzing a biomolecule analyte comprising the stepsof: a. preparing the biomolecule analyte using the method of claim 10;b. providing an apparatus having a first fluid chamber, a second fluidchamber, a membrane positioned between the first and second chambers anda nanopore extending through the membrane such that the first and secondchambers are in fluid communication via the nanopore; c. introducing thebiomolecule analyte into the first chamber; d. translocating thebiomolecule analyte from the first chamber through the nanopore and intothe second chamber; e. monitoring changes in an electrical propertyacross the nanopore as the biomolecule analyte is translocatedtherethrough, the changes in the electrical property corresponding tolocations along the biomolecule analyte containing probes; and f.recording the changes in the electrical property as a function of time.33. A method for analyzing a biomolecule analyte comprising the stepsof: a. preparing the biomolecule analyte using the method of claim 15;b. providing an apparatus having a first fluid chamber, a second fluidchamber, a membrane positioned between the first and second chambers anda nanopore extending through the membrane such that the first and secondchambers are in fluid communication via the nanopore; c. introducing thebiomolecule analyte into the first chamber; d. translocating thebiomolecule analyte from the first chamber through the nanopore and intothe second chamber; e. monitoring changes in an electrical propertyacross the nanopore as the biomolecule analyte is translocatedtherethrough, the changes in the electrical property corresponding tolocations along the biomolecule analyte containing probes; and f.recording the changes in the electrical property as a function of time.34. A method for analyzing a biomolecule analyte comprising the stepsof: a. preparing the biomolecule analyte using the method of claim 24;b. providing an apparatus having a first fluid chamber, a second fluidchamber, a membrane positioned between the first and second chambers anda nanopore extending through the membrane such that the first and secondchambers are in fluid communication via the nanopore; c. introducing thebiomolecule analyte into the first chamber; d. translocating thebiomolecule analyte from the first chamber through the nanopore and intothe second chamber; e. monitoring changes in an electrical propertyacross the nanopore as the biomolecule analyte is translocatedtherethrough, the changes in the electrical property corresponding tolocations along the biomolecule analyte containing probes; and f.recording the changes in the electrical property as a function of time.35. A method for analyzing a biomolecule analyte comprising the stepsof: a. preparing the biomolecule analyte using the method of claim 1; b.disposing the biomolecule analyte in a fluidic nanochannel ormicrochannel; c. applying a potential along the fluidic channel; d.translocating the biomolecule analyte from a first end of the fluidicchannel to a second end of the fluidic channel; and e. detectingelectrical properties as the biomolecule analyte moves through thefluidic channel, the electrical properties corresponding to at least onedetector volume in the fluidic channel, each detector volume beingdefined by two or more sensing electrodes disposed along the length ofthe fluidic channel, wherein the detected electrical signals indicatelocations of hybridized probes along the biomolecule analyte.
 36. Amethod for analyzing a biomolecule analyte comprising the steps of: a.preparing the biomolecule analyte using the method of claim 10; b.disposing the biomolecule analyte in a fluidic nanochannel ormicrochannel; c. applying a potential along the fluidic channel; d.translocating the biomolecule analyte from a first end of the fluidicchannel to a second end of the fluidic channel; and e. detectingelectrical properties as the biomolecule analyte moves through thefluidic channel, the electrical properties corresponding to at least onedetector volume in the fluidic channel, each detector volume beingdefined by two or more sensing electrodes disposed along the length ofthe fluidic channel, wherein the detected electrical signals indicatelocations of hybridized probes along the biomolecule analyte.
 37. Amethod for analyzing a biomolecule analyte comprising the steps of: a.preparing the biomolecule analyte using the method of claim 15; b.disposing the biomolecule analyte in a fluidic nanochannel ormicrochannel, c. applying a potential along the fluidic channel; d.translocating the biomolecule analyte from a first end of the fluidicchannel to a second end of the fluidic channel; and e. detectingelectrical properties as the biomolecule analyte moves through thefluidic channel, the electrical properties corresponding to at least onedetector volume in the fluidic channel, each detector volume beingdefined by two or more sensing electrodes disposed along the length ofthe fluidic channel, wherein the detected electrical signals indicatelocations of hybridized probes along the biomolecule analyte.
 38. Amethod for analyzing a biomolecule analyte comprising the steps of: a.preparing the biomolecule analyte using the method of claim 24; b.disposing the biomolecule analyte in a fluidic nanochannel ormicrochannel; c. applying a potential along the fluidic channel; d.translocating the biomolecule analyte from a first end of the fluidicchannel to a second end of the fluidic channel; and e. detectingelectrical properties as the biomolecule analyte moves through thefluidic channel, the electrical properties corresponding to at least onedetector volume in the fluidic channel, each detector volume beingdefined by two or more sensing electrodes disposed along the length ofthe fluidic channel, wherein the detected electrical signals indicatelocations of hybridized probes along the biomolecule analyte.